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A Small-Volume, Low-Cost, and Versatile Continuous Culture Device.

Matteau D, Baby V, Pelletier S, Rodrigue S - PLoS ONE (2015)

Bottom Line: However, commercially available instruments are expensive, were not designed to handle small volumes in the milliliter range, and can lack the flexibility required for the diverse experimental needs found in several laboratories.Furthermore, the selected light-emitting diode and photodetector enable the use of phenol red as a pH indicator, which can be used to indirectly monitor the bulk metabolic activity of a cell population rather than the turbidity.This affordable and customizable system will constitute a useful tool in many areas of biology such as microbial ecology as well as systems and synthetic biology.

View Article: PubMed Central - PubMed

Affiliation: Département de biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT

Background: Continuous culture devices can be used for various purposes such as establishing reproducible growth conditions or maintaining cell populations under a constant environment for long periods. However, commercially available instruments are expensive, were not designed to handle small volumes in the milliliter range, and can lack the flexibility required for the diverse experimental needs found in several laboratories.

Methodology/principal findings: We developed a versatile continuous culture system and provide detailed instructions as well as a graphical user interface software for potential users to assemble and operate their own instrument. Three culture chambers can be controlled simultaneously with the proposed configuration, and all components are readily available from various sources. We demonstrate that our continuous culture device can be used under different modes, and can easily be programmed to behave either as a turbidostat or chemostat. Addition of fresh medium to the culture vessel can be controlled by a real-time feedback loop or simply calibrated to deliver a defined volume. Furthermore, the selected light-emitting diode and photodetector enable the use of phenol red as a pH indicator, which can be used to indirectly monitor the bulk metabolic activity of a cell population rather than the turbidity.

Conclusions/significance: This affordable and customizable system will constitute a useful tool in many areas of biology such as microbial ecology as well as systems and synthetic biology.

No MeSH data available.


Related in: MedlinePlus

Calibration of the Versatile continuous culture device (VCCD) using batch cultures.(A) Comparison of 560nm transmittance measured by the VCCD and 600nm absorbance measured by a conventional spectrophotometer of an E. coli culture in LB broth. (B) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an E. coli culture grown in LB broth. Red squares are excluded from the correlation determination because they are not part of the exponential growth phase. (C) Relationship between relative 560nm transmittance measured by the VCCD and cell density of S. cerevisae growing in YPAD 2% glucose medium. (D) Relative 560nm transmittance of ATCC 1161 medium through different pH values generally observed during M. florum growth. (E) Cell density of M. florum growing in ATCC 1161 medium through pH decrease. (F) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an M. florum culture grown in ATCC 1161 medium.
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pone.0133384.g003: Calibration of the Versatile continuous culture device (VCCD) using batch cultures.(A) Comparison of 560nm transmittance measured by the VCCD and 600nm absorbance measured by a conventional spectrophotometer of an E. coli culture in LB broth. (B) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an E. coli culture grown in LB broth. Red squares are excluded from the correlation determination because they are not part of the exponential growth phase. (C) Relationship between relative 560nm transmittance measured by the VCCD and cell density of S. cerevisae growing in YPAD 2% glucose medium. (D) Relative 560nm transmittance of ATCC 1161 medium through different pH values generally observed during M. florum growth. (E) Cell density of M. florum growing in ATCC 1161 medium through pH decrease. (F) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an M. florum culture grown in ATCC 1161 medium.

Mentions: In order to use the VCCD to keep cell populations at a constant density for long periods, we first verified that the instrument could accurately measure the growth of different model microorganisms. Using the VCCD to monitor the growth of an E. coli BW25113 [25] batch culture in LB broth, we observed a decreasing 560nm transmittance signal as cells grew (S9A and S10A Figs), and more importantly, we noticed a strong correlation according to the Beer-Lambert law between 560nm transmittance values acquired by our system and 600nm absorbance measured by a conventional spectrophotometer (Fig 3A). This suggests that transmittance quantification by our LED-PHR approach offers performances comparable to commercially available instruments, even for measurements performed under ambient light. We also observed a strong linear correlation between log of cell concentrations and relative 560nm transmittance ranging from approximately 107 to 108 CFU/mL and 70% to 25%, respectively (Fig 3B). This range of transmittance roughly corresponds to an OD600nm signal between 0.2 and 0.5 (Fig 3A), an optical density interval typically associated with E. coli exponential growth phase. A similar pattern was also observed with S. cerevisae growing in YPAD medium with 2% glucose (Fig 3C, S10C and S10D Fig), clearly showing that the selected LED and PHR for the VCCD are suitable to follow the growth of commonly studied microorganisms.


A Small-Volume, Low-Cost, and Versatile Continuous Culture Device.

Matteau D, Baby V, Pelletier S, Rodrigue S - PLoS ONE (2015)

Calibration of the Versatile continuous culture device (VCCD) using batch cultures.(A) Comparison of 560nm transmittance measured by the VCCD and 600nm absorbance measured by a conventional spectrophotometer of an E. coli culture in LB broth. (B) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an E. coli culture grown in LB broth. Red squares are excluded from the correlation determination because they are not part of the exponential growth phase. (C) Relationship between relative 560nm transmittance measured by the VCCD and cell density of S. cerevisae growing in YPAD 2% glucose medium. (D) Relative 560nm transmittance of ATCC 1161 medium through different pH values generally observed during M. florum growth. (E) Cell density of M. florum growing in ATCC 1161 medium through pH decrease. (F) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an M. florum culture grown in ATCC 1161 medium.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4510131&req=5

pone.0133384.g003: Calibration of the Versatile continuous culture device (VCCD) using batch cultures.(A) Comparison of 560nm transmittance measured by the VCCD and 600nm absorbance measured by a conventional spectrophotometer of an E. coli culture in LB broth. (B) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an E. coli culture grown in LB broth. Red squares are excluded from the correlation determination because they are not part of the exponential growth phase. (C) Relationship between relative 560nm transmittance measured by the VCCD and cell density of S. cerevisae growing in YPAD 2% glucose medium. (D) Relative 560nm transmittance of ATCC 1161 medium through different pH values generally observed during M. florum growth. (E) Cell density of M. florum growing in ATCC 1161 medium through pH decrease. (F) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an M. florum culture grown in ATCC 1161 medium.
Mentions: In order to use the VCCD to keep cell populations at a constant density for long periods, we first verified that the instrument could accurately measure the growth of different model microorganisms. Using the VCCD to monitor the growth of an E. coli BW25113 [25] batch culture in LB broth, we observed a decreasing 560nm transmittance signal as cells grew (S9A and S10A Figs), and more importantly, we noticed a strong correlation according to the Beer-Lambert law between 560nm transmittance values acquired by our system and 600nm absorbance measured by a conventional spectrophotometer (Fig 3A). This suggests that transmittance quantification by our LED-PHR approach offers performances comparable to commercially available instruments, even for measurements performed under ambient light. We also observed a strong linear correlation between log of cell concentrations and relative 560nm transmittance ranging from approximately 107 to 108 CFU/mL and 70% to 25%, respectively (Fig 3B). This range of transmittance roughly corresponds to an OD600nm signal between 0.2 and 0.5 (Fig 3A), an optical density interval typically associated with E. coli exponential growth phase. A similar pattern was also observed with S. cerevisae growing in YPAD medium with 2% glucose (Fig 3C, S10C and S10D Fig), clearly showing that the selected LED and PHR for the VCCD are suitable to follow the growth of commonly studied microorganisms.

Bottom Line: However, commercially available instruments are expensive, were not designed to handle small volumes in the milliliter range, and can lack the flexibility required for the diverse experimental needs found in several laboratories.Furthermore, the selected light-emitting diode and photodetector enable the use of phenol red as a pH indicator, which can be used to indirectly monitor the bulk metabolic activity of a cell population rather than the turbidity.This affordable and customizable system will constitute a useful tool in many areas of biology such as microbial ecology as well as systems and synthetic biology.

View Article: PubMed Central - PubMed

Affiliation: Département de biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT

Background: Continuous culture devices can be used for various purposes such as establishing reproducible growth conditions or maintaining cell populations under a constant environment for long periods. However, commercially available instruments are expensive, were not designed to handle small volumes in the milliliter range, and can lack the flexibility required for the diverse experimental needs found in several laboratories.

Methodology/principal findings: We developed a versatile continuous culture system and provide detailed instructions as well as a graphical user interface software for potential users to assemble and operate their own instrument. Three culture chambers can be controlled simultaneously with the proposed configuration, and all components are readily available from various sources. We demonstrate that our continuous culture device can be used under different modes, and can easily be programmed to behave either as a turbidostat or chemostat. Addition of fresh medium to the culture vessel can be controlled by a real-time feedback loop or simply calibrated to deliver a defined volume. Furthermore, the selected light-emitting diode and photodetector enable the use of phenol red as a pH indicator, which can be used to indirectly monitor the bulk metabolic activity of a cell population rather than the turbidity.

Conclusions/significance: This affordable and customizable system will constitute a useful tool in many areas of biology such as microbial ecology as well as systems and synthetic biology.

No MeSH data available.


Related in: MedlinePlus